Archives of Virology (2021) 166:2627–2632 https://doi.org/10.1007/s00705-021-05167-y

ANNOTATED SEQUENCE RECORD

Novel (family Picornaviridae) from freshwater fshes (Perca fuviatilis, Sander lucioperca, and Ameiurus melas) in Hungary

Renáta Hargitai1 · Péter Pankovics1 · Ákos Boros1 · Róbert Mátics2 · Eda Altan3 · Eric Delwart3,4 · Gábor Reuter1

Received: 1 March 2021 / Accepted: 21 May 2021 / Published online: 13 July 2021 © The Author(s) 2021

Abstract In this study, a novel picornavirus (perchPV/M9/2015/HUN, GenBank accession no. MW590713) was detected in eight (12.9%) out of 62 faecal samples collected from three (Perca fuviatilis, Sander lucioperca, and Ameiurus melas) out of 13 freshwater fsh species tested and genetically characterized using viral metagenomics and RT-PCR methods. The complete of perchPV/M9/2015/HUN is 7,741 nt long, excluding the poly(A) tail, and has the genome organization 5’UTR​ IRES-?/ NPG↓P H-box/NC VPg Pro Pol P1(VP0-VP3-VP1)/P2(2A1 -2A2 -2B-2C)/P3(3A-3B -3C -3D )/3’UTR-poly(A). The P1, 2C, and 3CD proteins had 41.4%, 38.1%, and 47.3% amino acid sequence identity to the corresponding proteins of Wenling lepidotrigla picornavirus (MG600079), eel picornavirus (NC_022332), and Wenling pleuronectiformes picornavirus (MG600098), respectively, as the closest relatives in the genus Potamipivirus. PerchPV/M9/2015/HUN represents a potential novel fsh- origin species in an unassigned genus in the family Picornaviridae.

Introduction have also been identifed in lower vertebrates, including amphibians (newts) [16, 19], reptiles (tortoises) Picornaviruses are small non-enveloped with a pos- [8, 15], and fsh. In the last group, six ofcial fsh-origin itive-sense, single-stranded RNA genome, belonging to the picornavirus genera – Limnipivirus from bluegill [4], carp family Picornaviridae. The family consists of 158 species [13], and minnow [18]; Potamipivirus from eel [9] and stick- grouped into 68 genera (https://​www.​picor​navir​idae.​com/​ leback [12]; Fipivirus from sharpbelly, crossorhombus, jack index.​html). Not only is the known genetic diversity of the mackerel, wrasse, and banjofsh [20]; Symapivirus from tri- picornaviruses expanding rapidly but the number of known plecross lizardfsh [20]; Rajidapivirus from sharpspine skate host species is also increasing. Picornaviruses have been [20], and Danipivirus from zebrafsh [1] – have been estab- identifed in a wide range of vertebrates from fsh to mam- lished and approved (https://talk.​ ictvo​ nline.​ org/​ ictv-​ repor​ ts/​ ​ mals, including humans. Most of the known picornaviruses ictv_​online_​report/​posit​ive-​sense-​-​​es/w/​picor​navir​ were found in mammals and birds, but in recent years, novel idae.https://www.​ picor​ navir​ idae.​ com/​ index.​ html​ ), although there are further unassigned picornaviruses that have been discovered in diferent fsh species [20]. Handling Editor: Kalpana Agnihotri. Some picornaviruses are important viral pathogens, caus- ing a wide range of diseases in humans and wild, domestic, Nucleotide sequence data reported are available in the DDBJ/ and laboratory animals. However, little is known about the EMBL/GenBank databases under the accession number MW590713. diversity, epidemiology, and disease-causing potential of fsh picornaviruses [14]. * Gábor Reuter In this study, we report the identifcation and complete [email protected] genome characterization of a potentially novel picornavirus Perca fuviatilis, Sander lucioperca 1 Department of Medical Microbiology and Immunology, in freshwater fshes ( , and Medical School, University of Pécs, Szigeti út 12., Ameiurus melas) in Hungary. 7624 Pecs, Hungary 2 Hungarian Nature Research Society, Ajka, Hungary 3 Vitalant Research Institute, San Francisco, CA, USA 4 University of California, San Francisco, CA, USA

Vol.:(0123456789)1 3 2628 R. Hargitai et al.

Materials and methods were tested individually by the RT-PCR method, using the PerchPV-screen2-F/R (Supplementary Table S2) screening A total of 62 faecal samples were collected directly from primer pair. 13 diferent freshwater fsh (Supplementary Table S1) Sequence alignments of the P1, 2C, and 3CD regions living in natural and artifcial open-air fshponds in the were done using GeneDoc (version 2.7) and the MUSCLE vicinity of Szarvas (Eastern Hungary) in the year 2015. web server based on the amino acid sequences of the studied The fsh showed no clinical signs of disease during sample fsh picornavirus and representative picornaviruses available collection and were released immediately after sampling. in the GenBank database. Phylogenetic analysis was per- A specimen pool containing faecal samples from three formed using MEGA X (version 10.2.3), creating maximum- European perch (M9, M13 and M15) were selected for likelihood trees using the substitution model LG+F+G+I viral metagenomics analysis. Briefly, 200 μl of PBS- for P1 and 3CD analysis and LG+G+I for 2C, with 1000 diluted specimen was passed through a 0.45-μm sterile bootstrap replicates as described previously [1]. Potential flter (Millipore) and centrifuged at 6,000 × g for 5 min. secondary RNA structures of the 5’ and 3’ UTRs of the The fltrate was then treated with a mixture of DNases and analyzed picornavirus sequences were predicted using Mfold RNases (Turbo DNase, Invitrogen; Baseline Zero DNase, software [22]. Epicentre Biotechnologies; Benzonase Nuclease, Nova- The nucleotide (nt) and amino acid (aa) sequences of gen; RNase A, Fermentas) at 37° C for 2 hours to digest European perch picornavirus (perchPV/M9/2015/HUN) unprotected nucleic acids [17]. Viral-particle-protected have been deposited in the GenBank database under the nucleic acids were extracted using a MagMAX Viral RNA accession number MW590713. Isolation Kit (Ambion, Austin, USA) with RNase inhibitor (RiboLock RNase Inhibitor, Fermentas) during the elution step. Sequence-independent random RT-PCR amplifca- Results tion [21] with 20 PCR cycles was used, and a viral cDNA library with 250 base-paired ends was constructed using A specimen pool containing three faecal samples from Euro- a Nextera XT DNA Library Preparation Kit (Illumina). pean perch was subjected to viral metagenomics analysis. The library was sequenced on an Illumina MiSeq platform After de novo assembly of the 16,612,456 sequence reads, according to the manufacturer’s instructions. The metagen- 58,855 reads from this pool were found to show similarity -10 omic reads were trimmed, assembled de novo [7], and ana- (BLASTx cutof E-value ≤ ­10 ) to viral sequences. The lyzed using an in-house pipeline [17]. Briefy, the singlets sequences with more than 100 reads were from unclassifed and assembled contigs greater than 250 bp were compared viruses (n = 13,542), as well as the families to the GenBank [6] protein database using BLASTx (ver- (n = 15,209), (n = 12,081), (n sion 2.2.7) [2], using an E-value cutof of 0.01. Candidate = 5,360), Picornaviridae (n = 3,198), (n = viral hits were then compared to a non-viral non-redun- 2,689), (n = 1,825), (n = 794), dant protein database to remove false positive viral hits. (n = 643), (n = 289), Virus-family-level categorization of all viral metagenomic (n = 170), and (n = 133). sequences was based on the best BLASTx scores (E-value Sequence reads/contigs corresponding to family Picor- ≤ ­10-10). Metagenomic raw sequence read data are avail- naviridae were selected for further analysis. The longest able upon request. picornavirus contig was 3,423 nucleotides (nt) long, and A sequence-specifc screening primer pair (PerchPV- the encoded protein had only 40.2% aa sequence identity screen1-F/R, Supplementary Table S2) was designed for to the 2C/3C-3D region of eel picornavirus 1 strain F15/5 the 3D region to identify the viral genome of the study (NC_022332), a member of the species Potamipivirus A in strain from the specimen pool. The PCR thermocycler the genus Potamipivirus [9]. A sequence-specifc screening program consisted of 3 min at 95º C, 40 cycles for 30 primer pair was designed based on the 3D region of the s at 95º C, 20 s at 55º C, and 30 s at 72º C, and a fnal picornavirus sequence contig (Supplementary Table S2) to 3-min extension at 72º C using a C1000 Touch Thermal identify the picornavirus strain in the specimen pool. One Cycler (Bio-Rad). In addition, diferent sets of specifc of the three specimens from European perch was RT-PCR primers (Supplementary Table S2) were designed based positive, and this sample (M9) was selected for further study. on the sequences of the metagenomics reads/contigs and The complete genome – including the complete cod- the amplifed PCR products for the verifcation of the ing regions – of European perch picornavirus (perchPV/ metagenomics contig and to obtain the complete viral M9/2015/HUN, MW590713) is 7,741 nt long excluding the genome sequence, including the 5’ and 3’ ends, of the poly(A) tail (Fig. 1). The genome organization is as follows: IRES-?/ NPG↓P H-box/NC study strain (perchPV/M9/2015/HUN). All faecal samples 5’UTR​ P1(VP0-VP3-VP1)/P2(2A1 -2A2 - 2B-2C)/P3(3A-3BVPg-3CPro-3DPol)/3’UTR-poly(A). It has

1 3 Novel picornavirus from freshwater fshes 2629

Fig. 1 Schematic genome organization of perch picornavirus strain P4/P4’ cleavage sites are indicated above the genome map. Question perchPV/M9/2015/HUN (GenBank accession no. MW590713). The marks and dotted lines indicate the uncertain borders of VP3/VP1, genome map is drawn to scale. VP0, VP3, and VP1 represent viral VP1/2A1, and 2C/3A. The IRES type of the 5’UTR is unknown (see structural proteins and are shown in grey. Nucleotide (upper num- Fig. 2). aa, amino acid; nt, nucleotide; RdRp, RNA-dependent RNA ber) and amino acid (lower number) lengths are indicated in each polymerase; UTR, untranslated region; VP, viral protein gene box. Conserved picornaviral amino acid motifs and predicted

44.4% G+C content and a nucleotide composition of 31.6% 43.05% (coverage: 92%), and 38.79% (coverage: 88%) to the A, 24% U, 23.4% G, and 21% C. No nucleotide tandem corresponding proteins of Wenling lepidotrigla picornavirus repeats were found in the genome sequence. (MG600079), eel picornavirus strain F15/05 (NC_022332), The European perch picornavirus 5’UTR is 603 nt long. and Wenling lepidotrigla picornavirus (MG600079), respec- The frst in-frame AUG initiation codon is at nt position tively, using GenBank BLASTp. The complete poly-

604-606 in an optimal Kozak context (CACAA​ 604UGG, start protein (P1) shows the highest sequence identity (41.4%; codon in bold). Using a GenBank BLASTn search, a unique, coverage: 96%) to the corresponding polyprotein of Wenling 90-nt-long region with partial genetic similarity was found lepidotrigla picornavirus (MG600079). In the non-structural in the 5’UTR region of potamipivirus B1 (species Potamipi- region, two potential 2A proteins were predicted: the 19-aa- NPG↓P H-box/NC virus B, MK189163) from stickleback [12]. This nucleotide long ­2A1 and the 137-aa-long ­2A2 (Fig. 1). The region (between nt position 374 and 463 of the 5’UTR of 2C possesses the GxxGxGKS (G­ 1174PAGCGKS) motif for European perch picornavirus) has 81% nt sequence identity nucleotide triphosphate binding and the D­ 1237DMGQ motif to the corresponding non-coding region of potamipivirus for putative activity [11]. The 2C protein shows B1 (Fig. 2). The secondary RNA structure of the 5’UTR the highest aa sequence identity (38.1%, coverage: 94%) to including the IRES is unknown in potamipiviruses. Based the corresponding protein of eel picornavirus (NC_022332). on the 5’UTR sequences of European perch picornavirus The picornavirus 3C protease catalytic triad and well-con- and the above 5’UTR sequences of potamipivirus B1, the served aa motifs of 3D (RNA-dependent RNA polymerase) construction of a predicted RNA secondary structure model are present in the study strain (Fig. 1) [3, 10]. The 3CD pro- was partially successful (Fig. 2). The sequence alignment teins show the highest aa sequence identity (47.3%, cover- showed that the majority of the nucleotide mutations main- age: 99%) to the 3CD proteins of Wenling pleuronectiformes tained their base pairing (and therefore the predicted second- picornavirus (MG600098) from fatfsh [20]. ary RNA structure) in the aligned region regardless of the The 3’UTR is 148 nt long. Partial nucleotide sequence Watson-Crick or wobble nature of the base pairing. How- similarity (84-91% identity) was found between nt posi- ever, none of the fve known picornavirus IRES structures tion 7,612 and 7,679 of the 3’UTR of European perch (types I-V) or the known conserved 5’UTR-IRES nt motifs picornavirus to the 3’UTR regions of fve picornaviruses, [5] could be identifed in the new sequence. including Wenling lepidotrigla picornavirus (MG600079), The predicted length of the coding region (ORF) is 6,990 Yancheng osbecks grenadier anchovy picornavirus nt, and it encodes a single 2,329-aa-long polyprotein (Fig. 1). (MG600099), potamipivirus A (MK189163), eel picorna- The predicted capsid proteins (VP0, VP3, and VP1) had the virus (KC843627), and Wenling hoplichthys picornavirus highest aa sequence identity of 51.79% (coverage: 99%), (MG600101), using GenBank BLASTn search (Fig. 2). All

1 3 2630 R. Hargitai et al.

Fig. 2 Predicted RNA secondary structure of the 5’UTR (left side; HUN and potamipivirus B1 (MK189163) were compared. In the partial) and 3’UTR (right side; complete) of the genome sequence 3’UTR, the stem-loop structure at the apex (indicated by a line) is of perchPV/M9/2015/HUN. Signifcant nucleotide sequence similar- highly conserved (92-100% identity) in perchPV/M9/2015/HUN and ity to other fsh-origin picornaviruses, identifed using BLASTn, are fve other fsh-origin picornavirus nucleotide sequences. The protein highlighted with a grey background. In the 5’UTR, the majority of coding region extends from nucleotides 604 to 7593 in perchPV/ the nucleotide mutations maintained their base pairing (and therefore M9/2015/HUN in a single open reading frame (ORF). the predicted secondary RNA structures) when perchPV/M9/2015/

of these picornaviruses were identifed in diferent fsh Discussion species. Especially high (92-100%) nt sequence identity was found within the region between nt position 7,637 Since the frst confrmed report in 2014 [4], accumulating and 7,661 of the 3’UTR of European perch picornavirus data have shown that fsh are important vertebrate hosts (Fig. 2). We used these six 3’UTR nt sequences to predict of picornaviruses and therefore should not be neglected in the RNA secondary structure of the 3’UTR (Fig. 2). We picornaviral studies. Theoretically, and from an evolution- found that the majority of the nucleotide mutations main- ary point of view, lower vertebrates have been infected tained their base pairing (data not shown), and therefore, with a larger number of viruses and with genetically more the predicted RNA secondary structure, especially that of diverse viruses than mammals. At present, six ofcial fsh- the stem-loop at the apex is highly conserved in all of the origin genera of picornaviruses (Limnipivirus, Potamipi- sequences (Fig. 2). The 3’UTR has 64.2% A+U content. virus, Fipivirus, Symapivirus, Rajidapivirus, and Danipi- Phylogenetic analysis (Fig. 3) based on the amino acid virus) have been established, but several picornaviruses sequences of the P1, 2C, and 3CD proteins showed that identifed in saltwater and freshwater fshes are waiting perchPV/M9/2015/HUN represents a distinct lineage for their ofcial taxonomy to be determined [20]. This among picornaviruses, but it is clustered together with study reports the complete genome characterization and the main group of known but presently unassigned fsh molecular epidemiology of an additional novel picornavi- picornaviruses. rus from freshwater fsh. According to the current ICTV The prevalence of the novel picornavirus genome was Picornaviridae Study Group taxonomy guidelines (http://​ tested by RT-PCR in faecal samples from a total of 13 dif- www.picor​ nastu​ dygro​ up.​ com/​ defn​ itions/​ genus_​ defn​ ition.​ ​ ferent freshwater fsh species (Supplementary Table S1). A htm), one of the criteria for creating a new picornavirus total of eight (12.9%) of the 62 faecal samples from three genus is signifcant divergence of the orthologous protein fsh species were positive by RT-PCR for the study strain: sequences: exceeding 66% for P1 and 64% for 2C, 3C, three (37.5%) of eight, two (28.6%) of seven, and three and 3D. Based on complete genome sequencing and com- (75%) of four specimens from European perch, zander, and parative analysis, the strain reported here may represent black bullhead, respectively (Supplementary Table S1). a novel species and form a potential novel picornavirus Sequence analysis revealed that all of the sequences of genus in a populated monophyletic group of picornavi- 257-bp-long PCR products obtained using the PerchPV- ruses identifed in fsh. screen2-F/R primers were 100% identical to each other.

1 3 Novel picornavirus from freshwater fshes 2631

Fig. 3 Phylogenetic analysis of the (a) P1, (b) 2C, and (c) 3CD pro- 3CD protein sequences in the GenBank database. The P1 and 2C teins of prototype picornaviruses representing picornavirus genera trees show only selected fsh-origin picornavirus P1 and 2C protein and fsh-origin picornaviruses, including the study strain perchPV/ sequences, mainly those most closely related to the study strain. Phy- M9/2015/HUN (MW590713, bold letter and black arrows). Fish- logenetic trees were constructed in MEGA X, using the maximum- origin picornaviruses are indicated by black dots. The names of the likelihood method with the LG+F+G+I model for the P1 and 3CD ofcially accepted picornavirus genera of fsh origin are indicated. regions and the LG+I+G model for the 2C region. Bootstrap values The 3CD tree includes all of the available fsh-origin picornavirus (based on 1000 replicates) for each node are given if >50%.

Partial but signifcant nucleotide sequence similarity with wild, experimental, and farmed fsh bred for human was identifed in the 5’ and 3’ UTRs of perchPV/M9/2015/ consumption. HUN and fve other fsh-origin picornaviruses. The RNA secondary structure of these UTRs is not known, but they Supplementary Information The online version contains supplemen- tary material available at https://doi.​ org/​ 10.​ 1007/​ s00705-​ 021-​ 05167-y​ . could have a common origin. Based on their sequence simi- larity and presumed structural analogy, the partial 5’UTR Acknowledgements This work was supported by a grant from the Hun- and the complete 3’UTR secondary RNA structure could garian Scientifc Research Fund (OTKA/NKFIH K111615 and NKFIH be predicted. While the detailed 5’UTR structure, especially FK134311) and the PTE-ÁOK-KA No. 2020/14 of the Medical School, the type of the internal ribosome entry site (IRES) remains University of Pécs. P.P. was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. P.P. and B.Á. were unknown, the stem-loop structure in the apical part of the supported by the György Romhányi Research Scholarship of the Uni- 3’UTR is highly conserved in these virus sequences. versity of Pécs, Medical School. PerchPV/M9/2015/HUN was identifed in faecal speci- mens from diferent fsh living in geographically separated Funding Open access funding provided by University of Pécs. open-air fsh pond habitats. Whether this virus infects and Hungarian Scientifc Research Fund (OTKA/NKFIH K111615 and FK134311), Hungarian Nature Research Society and PTE ÁOK-KA replicates in specifc tissues in diferent fsh species or is able (No: 2020/14). to cause symptomatic infections remains to be determined. Investigation of the virome, including picornaviruses, Declarations of lower vertebrates contributes to our understanding of viral evolutionary relationships and allows the identifca- Conflict of interest The authors declare that they have no confict of tion of possible disease-causing picornaviruses associated interest.

1 3 2632 R. Hargitai et al.

Open Access This article is licensed under a Creative Commons Attri- chymotrypsin-like serine proteases: a distinct protein superfamily bution 4.0 International License, which permits use, sharing, adapta- with a common structural fold. FEBS Lett 243(2):103–114 tion, distribution and reproduction in any medium or format, as long 11. Gorbalenya AE, Koonin EV, Wolf YI (1990) A new superfamily as you give appropriate credit to the original author(s) and the source, of putative NTP-binding domains encoded by of small provide a link to the Creative Commons licence, and indicate if changes DNA and RNA viruses. FEBS Lett 262(1):145–148 were made. The images or other third party material in this article are 12. Hahn MA, Dheilly NM (2019) Genome characterization, preva- included in the article’s Creative Commons licence, unless indicated lence and transmission mode of a novel picornavirus associated otherwise in a credit line to the material. If material is not included in with the threespine stickleback fsh (Gasterosteus aculeatus). J the article’s Creative Commons licence and your intended use is not Virol 93:e02277-e2318 permitted by statutory regulation or exceeds the permitted use, you will 13. Lange J, Groth M, Fichtner D, Granzow H, Keller B, Walther M, need to obtain permission directly from the copyright holder. To view a Platzer M, Sauerbrei A, Zell R (2014) Virus isolate from carp: copy of this licence, visit http://creat​ iveco​ mmons.​ org/​ licen​ ses/​ by/4.​ 0/​ . genetic characterisation reveals a novel picornavirus with two 2A-like sequences. J Gen Virol 95:80–90 14. Mor SK, Phelps NBD (2016) Picornaviruses of fsh. In: Kibenge FSB, Godoy MG (eds) Chapter 21, Agriculture virology. Aca- References demic Press, pp 337–348 15. Ng TF, Wellehan JF, Coleman JK, Kondov NO, Deng X, Waltzek 1. Altan E, Kubiski SV, Boros Á, Reuter G, Sadeghi M, Deng X, TB, Reuter G, Knowles NJ, Delwart E (2015) A tortoise-infecting Creighton EK, Crim MJ, Delwart E (2019) A Highly divergent picornavirus expands the host range of the family Picornaviridae. picornavirus infecting the gut epithelia of zebrafsh (Danio rerio) Arch Virol 160(5):1319–1323 in research institutions worldwide. Zebrafsh 16(3):291–299 16. Pankovics P, Boros Á, Tóth Z, Phan TG, Delwart E, Reuter G 2. Altschul SF, Madden TL, Schäfer AA, Zhang J, Zhang Z, Miller (2017) Genetic characterization of a second novel picornavirus W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new from an amphibian host, smooth newt (Lissotriton vulgaris). Arch generation of protein database search programs. Nucleic Acids Virol 162:1043–1050 Res 25:3389–3402 17. Phan TG, Vo NP, Boros Á, Pankovics P, Reuter G, Li OT, Wang 3. Argos P, Kamer G, Nicklin MJ, Wimmer E (1984) Similarity C, Deng X, Poon LL, Delwart E (2013) The viruses of wild pigeon in gene organization and homology between proteins of animal droppings. PLoS ONE 8(9):e72787 picornaviruses and a plant suggest common ancestry 18. Phelps NBD, Mor SK, Armien AG, Batts W, Goodwin AE, Hop- of these virus families. Nucleic Acids Res 12(18):7251–7267 per L, McCann R, Ng TFF, Puzach C, Waltzek TB, Delwart E, 4. Barbknecht M, Sepsenwol S, Leis E, Tuttle-Lau M, Gaikowski Winton J, Goyal SM (2014) Isolation and molecular characteriza- M, Knowles NJ, Lasee B, Hofman MA (2014) Characterization tion of a novel picornavirus from baitfsh in the USA. PLoS ONE of a new picornavirus isolated from the freshwater fsh Lepomis 9(2):e87593 macrochirus. J Gen Virol 95(3):601–613 19. Reuter G, Boros Á, Tóth Z, Phan TG, Delwart E, Pankovics P 5. Belsham GJ (2009) Divergent picornavirus IRES elements. Virus (2015) A highly divergent picornavirus in the amphibian the Res 139(2):183–192 smooth newt (Lissotriton vulgaris). J Gen Virol 96:2607–2613 6. Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman 20. Shi M, Lin XD, Chen X, Tian JH, Chen LJ, Li K, Wang W, Eden DJ, Ostell J, Sayers EW (2013) GenBank. Nucleic Acids Res JS, Shen JJ, Liu L, Holmes EC, Zhang YZ (2018) The evolution- 41:D36-42 ary history of vertebrate RNA viruses. Nature 556:197–202 7. Deng X, Naccache SN, Ng T, Federman S, Li L, Chiu CY, Del- 21. Victoria JG, Kapoor A, Li L, Blinkova O, Slikas B, Wang C, wart E (2015) An ensemble strategy that signifcantly improves Naeem A, Zaidi S, Delwart E (2009) Metagenomic analysis of de novo assembly of microbial genomes from metagenomic next- viruses in stool samples from children with acute faccid paralysis. generation sequencing data. Nucleic Acids Res 43:e46 J Virol 83:4642–4651 8. Farkas SL, Ihász K, Fehér E, Bartha D, Jakab F, Gál J, Bányai 22. Zuker M (2003) Mfold web server for nucleic acid folding and K, Marschang RE (2015) Sequencing and phylogenetic analysis hybridization prediction. Nucleic Acids Res 31(13):3406–3415 identifes candidate members of a new picornavirus genus in ter- restrial tortoise species. Arch Virol 160:811–816 Publisher’s Note Springer Nature remains neutral with regard to 9. Fichtner D, Philipps A, Groth M, Schmidt-Posthaus H, Granzow jurisdictional claims in published maps and institutional afliations. H, Dauber M, Platzer M, Bergmann SM, Schrudde D, Sauerbrei A, Zell R (2013) Characterization of a novel picornavirus iso- late from a diseased European eel (Anguilla anguilla). J Virol 87(19):10895–10899 10. Gorbalenya AE, Donchenko AP, Blinov VM, Koonin EV (1989) Cysteine proteases of positive strand RNA viruses and

1 3